Injectable opacifying composition containing liposomes of high encapsulating capacity for X-ray examinations

ABSTRACT

Injectable aqueous composition intended for opacifying certain organs with a view to examination by X-rays. This composition is based on liposomes containing, encapsulated thereein, an aqueous solution of an iodinated opacifying agent. The ratio between the weight of iodine encapsulated by the liposomes and the weight of the lipids from which their membrane is formed is not lower than 1.5 mg/mg.

This is a continuation of application Ser. No. 07/302,690, filed Jan.19, 1989; now U.S. Pat. No. 5,312,615.

The present invention relates to an aqueous composition which can beinjected into the circulatory system of a patient and the purpose ofwhich is to opacify certain organs with a view to their diagnosticexamination with X-rays. This composition is formed of a suspension, ina physiologically acceptable aqueous medium, of liposomes as vesicleswith a phospholipidic membrane containing, encapsulated in thesevesicles, an aqueous solution of at least one iodinated compound whichis opaque to the X-rays.

It is known to use suspensions of liposomes as vehicles for thetransportation, in certain organs which are to be studied, of opacifyingagents intended for radioscopic examinations. Thus, the specification ofU.S. Pat. No. 4,192,859 describes such a suspension of liposomesconstituted of lecithin and sterols and containing about 20 to 60% byweight of a contrast agent intended for the examination of organs,particularly of organs in relation with the reticuloendothelial andcardiovascular systems, as well as lymphographic examinations. Amongsuch contrast agents, the following compounds are referred to in thisspecification:

N,N'-bis[2-hydroxy-1-(hydroxymethyl)-ethyl]-5-[(2-hydroxy-1-oxopropyl)-amino]-2,4,6-triiodo-1,3-benzene-dicarboxy-amide(iopamidol); metrizamide; diatrizoic acid; sodium diatrizoate; megluminediatrizoate; acetrizoic acid and its soluble salts; diprotrizoic acid;iodamide, sodium iodipamide, meglumine iodipamide, iodohippuric acid andthe soluble salts thereof; iodomethamic acid;iodopyracetiodo-2-pyridone-N-acetic acid, 3,5-diiodo-4-pyridone-N-aceticacid (Iodopyracet); the diethylammonium salt of the preceding acid;iothalmic acid; metrizoic acid and the salts thereof, the ipanoic,iocetamic, iophenoxic acids and their soluble salts; sodium tyropanoate,sodium ipodate and also other similar iodised compounds. The lipidicmembrane of the liposomes which are used in accordance with thisdocument mainly contains phospholipids, a sterol, locithin, dicetylphosphate or stearyl amine, and an organic solvent. Always in accordancewith this document, it is possible to prepare such liposomes by theselected lipidic components being mixed in a container with an organicsolvent, such as chloroform, dichloromethane, ethyl ether, carbontetrachloride, ethyl acetate, dioxane, THF, etc. After having evaporatedthe volatile compounds under reduced pressure, the lipidic mixture isdispersed in a buffering solution containing a measured quantity of anopacifying agent. The whole is then stirred for several hours, thiscausing the formation of liposomes, a part of the dispersion liquid(containing the opacifying agent) then being encapsulated in theliposomic vesicles which are thus produced. The dispersion is thensubjected to a sonication in order to reduce the size of these liposomesand the viscosity of the dispersion.

There are other documents which are concerned with the preparation ofliposomes which contain opacifying agents.

For example, the specification of FR-A-2,561,101 describes a process forthe preparation of liposomes which may contain X-ray opacifying agents.According to this specification, there are first of all prepared"precursors" of liposomes in organic solution, from which then thesolvent is partially separated, this solution containing a proportion oflipids which are adapted to convert the monomolecular membrane of theprecursors into bimolecular membrane. This solution is then dispersed inan aqueous medium and the residual solvent is completely eliminated.

The specification of U.S. Pat. No. 4,567,034 describes X-ray opacifiersand the incorporation thereof into the liposomes as vector of a contrastproduct.

The specification of GB-A-134,869 describes a technique for thepreparation of liposomes, in accordance with which particles (10 μm) ofa hydrosoluble carrier agent (NaCl, saccharose, lactose, etc.) arecoated with an amphipatic agent, the subsequent dissolution of thecarrier in an aqueous medium yielding the liposomes. The coating iseffected by dispersing the solid particles of the carrier in an organicsolution of the lipid and of the product to be encapsulated, among whichare found the X-contrast agents. Among the amphipatic agents, there areto be mentioned the saturated synthetic lecithins.

The specification of GB-A-2,135,268 also describes a process for formingliposomes which may contain opacifiers, starting from particles of ahydrosoluble carrier agent.

The specification of GB-A-2,135,647 describes a process for thepreparation of liposomes which are somewhat similar to that of the twopreceding documents, except for the difference that the particles of thecarrier material are insoluble. What are involved here are microspheresof glass or synthetic resin. These particles are coated with a filmcontaining a lipid and, optionally, an ionic surfactant or cholesterol,by bringing them into contact with an organic solution of theseingredients, the operation being followed by an evaporation. Thereafter,by agitating these spheres in an aqueous dispersion medium to beencapsulated, more particularly containing an X-ray opacifier, thenseparating out the latter by filtration or centrifugation, there isobtained the desired solution of liposomes.

The specification of GB-A-2,156,345 describes diacetylaminated compoundsof triiodobenzoic acid as X-contrast agents and the incorporationthereof into liposomes.

The specification of GB-A-2,157,283 describes compounds which aresimilar to those of the preceding document and the incorporationthereof, in amounts of 20-60%, into liposomes. The latter are inconformity with the preparation which is described in the specificationof U.S. Pat. No. 3,957,971.

The specification of EP-A-179,660 describes a process for preparingsuspensions of liposomes, in accordance with which a bioactive substanceto be encapsulated is dispersed in the presence of lipids in organicsolution, the solvent is evaporated until there is formation of a geland this latter is redispersed in a second buffering aqueous medium. Bya suitable control of the respective quantities of the ingredients andof the operating conditions, this results in a very high degree ofencapsulation and a very uniform distribution of the liposomic vesicles.

Among other recent documents which are concerned with liposomes, asvectors of contrast products, they may also be mentioned: A. HARVON etal. Radiology (1981) 140, 507; P. J. RYAN et al., Biochim. Biophys. Acta(1983), 756, 106; S. E. SELTZER et al., AJR (1984) 143, 575; P. J. RYANet al. , Radiology (1984) 152, 759; O. A. ROZENBERG Radiology (1983)149, 877; S. BENITA et al., J. Pharm. Sci (1984) 73, 1751; K. T. CHENGet al., Investigative Radiology (1987) 22, 47-55; M. R. ZALUTZKY et al.,investigative Radiology (1987) 22, 141-147.

Although the said art disclosed in the aforementioned documents performswell in experimental tests, certain practical problems as regardsutilisation remained to be resolved.

Thus, although the liposomic vesicles containing an opacifying agent arefinally fixed in the liver and the spleen, it is also possible for them,with their displacement in the circulatory system, to be retained by thecapillaries of the lungs with the risk of fatty embolism. Moreover, thespecific capacity of encapsulation by volume and weight of iodinatedproducts of the actual liposomes is relatively small (usually less than1 g of iodine per g of lipids), and this necessitates the injection of arelatively large quantity of lipids in order to achieve the desiredopacifying effect. On this subject, it is noted that, in practice, it iscustomary to characterize a preparation of liposomes by the amount ofencapsulated iodine as g of iodine per g of lipids (ratio I/l), althoughobviously this iodine is bonded to an organic molecule. As regards thequantity of iodine necessary for an examination using X-rays, it isadmitted that the opacification of the liver requires a concentration ofthe order of 2-2.5 mg of iodine per g of tissue, i.e. about 6 g (weightof liver about 2.3 kg). Taking account of the fact that only about 40%of the injected liposomes are retained in the liver, it is thereforenecessary for a minimum of 15 g of iodine to be administered. For aratio I/l of 1 (a high value in the state of the art) this correspondsto 15 g of lipids, which constitutes an already considerable dose. It isseen from this that the major interest is to increase the encapsulationcapacity of the liposomes.

By way of example, it is noted that, according to V. J. CARIDE, in CRCCritical Reviews in Therapeutic Drug Carrier Systems (1985), 1. 121-153,the encapsulation capacity of liposomes of the class which he defines as"small unilamellar vesicles, (SUV), which have a diameter between 0.02and 0.5 μm, is of the order of 0.2 to 1.5 l/mole, which corresponds,admitting a mean molecular weight of about 800 (phospholipids), to anencapsulation capacity of about 1 to 2 ml/g of phospholipids. Now it isdesirable to raise this capacity to 5 ml/g or more, if possible, so thatthe ratio I/l, for example, when encapsulting opacifying solutions ofthe order of 300 mg of iodine/ml, may exceed the value of 1.5.

Moreover, an appreciable part of the iodine which is contained by thepresent suspensions of liposomes with opacifying capacity is dissolvedin the aqueous dispersion phase and not encapsulated within the vesicles(see, for example, the suspensions described in the specification ofU.S. Pat. No. 4,192,859). Such a situation may be found to beundesirable because, at the time of injection for diagnostic purposes,the portion of non-encapsulated iodine is not fixed in the organs to beinvestigated and does not serve any useful purpose. Consequently, inorder to avoid having to inject iodine for nothing, it is desirable tobe able to decrease as far as possible this non-encapsulated fraction.

The composition as defined in the claim 1 permits the disadvantages oflack of encapsulation capacity to be overcome. Actually, during theirexperiments, the present inventors have established to their surprisethat, in "normalising" the size of the vesicules of the solution ofliposomes in a certain range of values, i.e. by eliminating, by sizingby extrusion, the major part of the vesicles with a dimension below 0.15μm or exceeding 3 μm, and preferably by keeping the major part of thevesicles at a size which is between 0.2 and 1 μm, the amount ofencapsulated iodine was increased in a significant manner. This effectis the result of an increase in the specific encapsulation capacity ofthe vesicles (i.e. the volume of encapsulated liquid in relation to theweight of the lipids of the vesicular membranes), it being possible, incertain cases for this ratio to reach 10-15 ml/g of lipid.

In addition, and constituting another unexpected element, the procedureof calibrating the liposomes has made it possible to obviate, to aconsiderable degree, the problem of the retention of the liposomes inthe capillaries of the lungs, the proportion of such liposomesdetectable in this organ decreasing considerably as soon as the largesize liposomes, for example, exceeding 2 to 3 μm, have been eliminated.It will be specified here that, by normalisation, it is wished to statethat, in accordance with the definitions relating to the standards ofstatistical distribution of the particles according to their size, theoperation of normalization leads to a contraction of the said vesicledistribution curve; thus, in the present invention, the index of theirpolydispersity is not higher than 4 and, preferably, the size of morethan 70% of the total number of vesicles of the solution of liposomes isbetween 0.2 and 2 μm. It will hereinafter be seen, in the sectionconcerned with the preparation techniques, what is to be understood bypolydispersivity index.

Because of the above discoveries and by an appropriate choice of theopacifying iodinated compounds dissolved in the encapsulated solutionaccording to claim 1, it has been possible successfully to obtain theopacifying compositions according to claims 2, 3 and 11. Moreover, bysubjecting such a solution to certain appropriate treatments (hereafterdescribed), a large part of the iodine dissolved in the aqueoussuspension phase has been successfully eliminated. It will also be notedthat the viscosity of the composition according to the invention, whichmay, in certain forms of execution, be lower than 30 or of the order of20 to 30 mP.s at 37° C., is clearly lower than that of the suspensionsaccording to the prior art (for instance, the suspensions described inthe specification of U.S. Pat. No. 4,192,859 may, for iodine contents of60%, reach viscosities of several hundreds of mP.s.). It is in factevident that the viscosity of the suspensions of liposomes decreaseswhen the lipid level diminishes and that, to keep the I/l ratioconstant, it is necessary to increase to the same extent theencapsulation capacity of these latter.

Furthermore, the presence of iodinated opacifying agents contributesalso to raising the viscosity of the solutions (for example, an aqueoussolution of iopamidol at 300 g of iodine per liter has a viscosity of8.8 mP.s at 20° C. and 4.7 mP.s at 37° C.) and any diminution of theconcentration of the iodinated compound in the dispersion medium of theliposomes will contribute to lowering this viscotiy.

The lipidic membrane of the liposomes of the composition according tothe invention may be constituted of the amphipatic compounds normallyemployed in the usual practice of liposomic suspensions. Liposomesemployed in the present invention generally comprise lipid materials,predominantly of the phospholipid type (for example, a sterol),lecithin, dioctyl phosphate, or stearylamine, in an organic solvent.Such compounds are described in the aforementioned references. It ispreferred to use phospholipids, such as the hydrogenated lecithins ofsoya (for example, the products NC-95H of Nattermann Chemie),dipalmitoyl phosphatidyl choline (DPPC), distearoyl phosphatidyl choline(DSPC), sphingomyeline (SM) dicetylphosphate (DCP),dipalmitoyl-phosphatidyl glycerol (DPPG) and dipalmitoyl phosphatidicacid (DPPA). Contrary to the usual practice in the sphere of liposomes(cf. the references cited in the introduction), it is preferred not touse cholesterol among the lipids employed in the present invention, thisnot being essential for their stabilisation. The proportion of lipidsrelatively to the dispersion buffering phase is generally or the orderof 0.1 to 10%, preferably about 2 to 6%. The proportion of encapsulatedliquid relatively to the lipids ( ratio v/l) is not below 5 ml/g and mayin exceptional cases reach 20 ml/g. Preferably, it is between 5 and 15ml/g, and most frequently between 7 and 12 ml/g.

As iodinated organic compounds which are opaque to X-rays, it ispossible to use most of the compounds known from the aforementionedreferences; however, certain opacifiers are more suitable than others asregards the effective encapsulation capacity of their aqueous solutionsin the liposomic vesicles and the stability of these vesicles instorage. Actually, certain of the opacifying agents in solution are moredifficultly encapsulated than others at the time of formation of theliposomes and, moreover, certain of them diffuse more easily than othersoutside the liposomic membrane, in storage or at the time of handling.

For these reasons, it is preferred to use, as opacifying agents, theionic contrast means derived from triiodobenzoic acid, such as, forexample, the sodium and/or meglumine salts of diatrizoic acid, andpreferably the non-ionic contrast means, such as iopamidol or iomeprol,given as a non-limiting example. The contrast agents are used in aqueoussolution form with a concentration which is between 100 and 450 g ofiodine/l, preferably of 250 to 380 gI/l. With such solutions, and thecomposition according to the invention, there are obtained amounts ofencapsulated iodine which may be up to 6 g of iodine/g of phospholipid.

In general, with the composition of the invention, the volume occupiedby the liposomic vesicles represents about 5 to 60% of the total volumeof the suspension and may, in certain special cases, exceed these values(up to 70-80%).

For preparing the composition according to the invention, i.e. forincreasing the encapsulation capacity of the liposomic vesicles and, forexample, to provide an aqueous suspension of liposomes, the vesicles ofwhich have a phospholipidic membrane with a power of encapsulating anopacifying aqueous liquid greater than 1.5 g of iodine per g of lipid(I/l>1.5), the procedure is to normalise the size of these vesicles,i.e. to select an important proportion of vesicles of which the size iscontained within a given range and eliminate the others, or by somemeans, to transform these latter (the others) into new vesicles havingdimensions which correspond to the chosen range. By the term "importantproportion" in respect of the normalisation of the size of theliposomes, reference is made to the notion of polydispersivity factor Pused when measuring the dimensions of particles and the distribution ofthese particles by diffraction spectroscopy (see instructions for usingthe COULTER Nano-Sizer apparatus (registered Trade Mark)--COULTERELECTRONICS LTD., Great Britain). The scale of the values of P rangesbetween 0 and 10. A value of 1 corresponds to monodimensional particles.A value of 8, for example, indicates that the ratio of the dimensionsbetween the largest and the smallest particles is about 4.

Moreover, the factor s, which permits to calculate the extent W of thedistribution curve of the particles, i.e. the dimension range of themajority of them, according to the relation W=sd_(w) (where d_(w) is thesize of the particles given by the apparatus) is provided by dividing Pby 5 for the particles larger than 250 nm and by 4 for particles between100 and 250 nm. In the present invention, reference is made to the valueof the polydispersivity index P and it is admitted that, for values of Pequal to or smaller than 4, the majority of the vesicles corresponds tothe measured size.

Therefore, the process as claimed in claim 4 illustrates a means forarriving at a composition such as defined in claim 1. It has in factbeen ascertained that these are the liposomes of which the majority ofthe vesicles have a size which is between about 0.15 and 3 μm,preferably 0.2 and 1 μm, which have a particularly high encapsulationcapacity. By the term "majority", it is wished to express the fact thatat least 70% of the liposomic vesicles have a diameter conforming to thechosen range.

Although the exact reason why the liposomic vesicles contained in thisrange have the capacity of integrating such a high volume has not beenelucidated, it is possible to advance the following arguments. Firstly,the liposomes below this limit have an unfavourable volume/surface ratio(actually, the more the diameter of a sphere decreases, the smaller thisratio becomes) and secondly, the vesicles exceeding approximately 2 μmare often plurilamellar and consequently, the mass of their membrane,for a given capacity, is higher. It appears that, by extrusion through acorrectly calibrated membrane, the plurilamellar liposomes arere-arranged, at least in part, into smaller liposomes having amonolamellar membrane; a large proportion of these "re-arranged"liposomes then corresponds to the optimal dimensions suitable for thecomposition according to the invention.

Generally, the normalisation of a solution of liposomes is effected bybeing forced under pressure through a filtering membrane. The pressuresbrought into play by this "extrusion" may vary between a fraction of abar and several bars. Preferably, for membranes having a porosity whichis between about 0.4 and 2 μm, extrusion pressures from 0.5 to 10 barare used. In this way, it is possible to assure filtration rates of theorder of 1 to 20 ml/sec/cm². It is quite understood that the increasingof the I/l ratio following the extrusion treatment is produced when theaqueous dispersion phase contains an iodinated opacifying compound in anappreciable proportion is evident that if this phase is deprived ofiodine, it is not possible to have an increase of the encapsulatediodine by passage of the exterior phase towards the interior of thevesicles.

It has been established that the extrusion temperature plays a part inconnection with the concentration of opacifying agents of the solutionencapsulated in the vesicles. Thus, if the extrusion is effected atnormal temperature, it is possible to produce a certain diminution ofthe I/l ratio. On the contrary, and this constitutes an addditionalunexpected element, if one proceeds at a temperature higher than thetransition temperature of the phospholipids forming the wall of theliposomes, there is observed an increase in this ratio. Preferably,temperatures between 50° and 90° are used, for example, in the region of75° C. It may be imagined that the unexpected result which is observedis due to a softening of the vesicles, caused by the raising of thetemperature.

For lowering as much as possible the quantity of non-encapsulated iodinecontained in the suspension, i.e. the portion of opacyfing agentdissolved in the buffering aqueous phase in which the liposomes aresuspended, ultracentrifugation or ultrafiltration have advantageouslybeen used, these procedures causing a physical separation between thevesicles themselves and the said aqueous phase. Once this separation isachieved, the liposomes are redispersed in a new aqueous dispersionphase. By repeating this operation, it is possible to reduce theproportion of opacifying agent in the exterior medium to a chosencontent, for example, of the order of 2 mg/ml or even 0.2 mg/ml, withoutthe loss of liposome (inevitable with each operation) becomingconsiderable. In general, such centrifuging operations are conductedwith centrifugal accelerations of several thousands of g, for example,between 10,000 and 250,000 g. It is also possible to obtain such resultsby microfiltration, or dialysis, in accordance with usual techniques. Itis possible to effect microfiltration operations by causing circulationof the suspension to be treated in a set of tubes of which the wall hasa determined controlled porosity, for example, pores of 0.1 μm and more(in the case of the previously mentioned ultrafiltration, the pores ofthe membranes are smaller than 0.1 μm). In proportion as the volume ofthe suspension subjected to the filtration decreases (by passage of apart of this suspension through the pores of the tubes), it is replacedby fresh solvent, for example, a buffering mixture or a physiologicallyacceptable aqueous solution. Using these techniques, most of theundesirable substances contained in the dispersion liquid, particularlythe dissolved iodine, are eliminated. Furthermore, the microfiltrationpermits eliminating, in the filtrate, certain undesirable solutions,especially the very small residual vesicles, this having the effect ofimproving the I/l ratio.

As media constituting the exterior suspension phase of the liposomes, itis possible to use solutions which are compatible with the livingtissues and the liquids of the circulatory system. To be mentioned asexamples of such solutions are the salt solutions, aqueous solutions,buffered or not with Tris, phosphate, etc. (pH in the region ofneutrality) and the hypertonic solutions containing one or moresubstances selected from salt, glucose, opacifying agents, bufferingagents, etc. One typical solution (0.8 Osm) contains glucose (0.7M),NaCl (0.9%) and Tris (10 mM).

For the preparation of suspensions of liposomes capable of being used asstarting products in the present invention, it is possible to use knowntechniques, particularly those described in the previously citedreferences.

Preferably used is the REV method. (cf. F. Szoka et al., (1978), Proc.Natl. Acad. Sci. USA 75, 4194) and that described in the specificationof EP-A-179.660.

By application of these methods, there are obtained initial suspensionsof liposomes which, in general, have the following parameters: buffer0.9% NaCl, 10 mM Tris, pH 7-7.5;

lipids, about 1%

total iodine concentration, 20-30 mg/ml;

iodine concentration in the liposomes 1-2 g/g of lipids (solution ofiopamidol at 300 g/l).

By subjecting such initial solutions to the aforementioned operations,there are obtained opacifying compositions in accordance with theinvention.

As the time of its use as opacifying agent by injection into laboratoryanimals, the composition according to the invention is shown to be veryeffective, on account of its specific opacifying power and itsselectivity. Particularly observed is a reduction by 30 to 40 times ofthe retention of iodine in the lungs. It is to be particularly notedthat, with the present composition, using a total amount by weight ofiodine smaller than 20%, it is possible to obtain diagnostic resultsequivalent or superior to those obtained with suspensions according tothe prior art (see, for example, U.S. Pat. No. 4,192,859), wherein theglobal concentration of iodine may reach 60% by weight of suspension ofliposomes.

The experimental part which follows illustrates the invention.

EXAMPLE 1

First of all, a solution is prepared which contains 57 mg of dipalmitoylphosphatidic acid (DPPA, Fluka) and 543 mg dipalmitoyl phosphatidylcholine (DPPC, Fluka) in 42 ml of chloroform. To 20 ml of this solutionare added 20 ml of chloroform and 40 ml of diisopropyl ether and then,after stirring, 12 ml of a 76% (p/v) aqueous solution of megluminediatrizoate, an iodinated opacifying agent (Bracco). The mixtureobtained, heated to 50° C., was subjected for 5 minutes to ultrasonics(Braun Labsonic 1510). The emulsion was then concentrated at 45° C. in arotary evaporator until a gel was obtained. A mixture of about 8 ml ofthe 76% aqueous solution of meglumine diatrizoate and 4 ml of distilledwater was then introduced into the flask and the evaporation wascontinued with rotation. After obtaining a homogeneous mixture, therewas again added a mixture of about 20 ml of the solution with 76% ofmeglumine diatrizoate and 8 ml distilled water and the last traces ofsolvents were eliminated by evaporation. The volume of the solution ofliposomes as obtained (solution A) was adjusted to 40 ml by means ofdistilled water.

The quantity of meglumine diatrizoate effectively encapsulated withinthe liposomes was then determined. An aliquot of the preparationobtained (5 ml) was centrifugated for 25 minutes at 235,000 g. Thevesicles were taken up in 10 ml of a saline solution (0.9% NaCl, 10 mMTrisHCl, pH 7.2) and then subjected to a second centrifugation operation(15 minutes, 26,000 g). These phases of centrifugation, followed bytaking up in suspension, were repeated another four times, this makingpossible the complete elimination of non-encapsulated megluminediatrizoate. After a last suspension in 5 ml, an aliquot of the solutionobtained (0.9 ml) was added to 0.1 ml of a 10% solution of sodiumdodecyl sulphate and then heated to 40° C. for 5 minutes. By measuringthe optical density at 260 nm of this solution, it was determined atthis stage that final preparation contained 21.4 mg/ml of megluminediatrizoate, corresponding to 10.4 mg of iodine per ml. By disregardingthe losses of lipids, it is established that this preparation wouldlikewise contain 7.14 mg/ml of phospholipids, i.e. aniodine/phospholipids ratio of 1.45.

The remainder of the solution A was brought to 75° C., then extrudedunder heat through a polycarbonate filter (Nuclepore) with a porosity of1 micron. The solution obtained was then cooled to ambient temperatureand thereafter subjected to a series of centrifugations, followed bybeing taken up in suspension, as described above. Spectrophotometricanalysis of the supernatant liquid obtained after the fifth washingshowed a residual iodine concentration in the external phase lower than0.2 mg/ml. At this stage, the residue of liposomes was suspended in atotal volume of 7 ml of buffer. In order to determine the totalconcentration of iodine in the final preparation, an aliquot quantity ofthis preparation was incubated, as described above, for 5 minutes at 40°C. in the presence of sodium dodecyl-sulphate. Spectrophotometricanalysis showed that the final preparation contained 128.5 mg/ml ofmeglumine diatrizoate corresponding to 62.5 mg of iodine per ml andpresenting an I/L ratio of 1.75. The extrusion has thus led to anincrease of 50% of the encapsulated iodine.

EXAMPLE 2

To 100 ml of diisopropyl ether were added 100 ml of a mixture ofphospholipids containing the following substances, in parts by weight:DPPC 3/DPPA 1/DSPC 1, this mixture being dissolved in chloroform at theconcentration (by weight) of 7.1 mg/ml.

There were then added 30 ml of a 61.2% aqueous solution of iopamidol(300 mg of iodine per ml) and the whole was then subjected for 6 minutesto a sonic treatment using ultrasonic waves at 50° (BRAUN Labsonic 1510ultrasonic apparatus). The milky solution was then evaporated, using theRotavapor (45°/8 mm Hg) in order to eliminate the volatile solvents. Thegel as formed was re-dispersed in 100 ml of the iopamidol solution.Samples of this preparation were then subjected to extrusion teststhrough (Nuclepore) membranes of 0.8 μm, 1 μm or 2 μm at 75° C. under apressure of about 5 bars.

The various extruded or non-extruded preparations were then subjected toultracentifugation (235,000 g; 30 minutes), after which the liposomicvesicles were re-dispersed in a buffering medium (0.9% NaCl 10 mM Tris,pH 7.2) (Tests 1 and 2). According to a modification, there was used, asdispersion phase, an iso-osmolar medium to the iopamidol solution, i.e.formed of 0.7M glucose, 15 mM NaCl, 1 mM Tris (test 3). Thispurification step (elimination of the iodine dissolved in the dispersivephase) was then repeated a certain number of times with one or other ofthese solutions, the centrifuging taking place at 26,000 g for 15minutes, down to a residual iodine content of the dispersion phase below0.2 mg/ml. This content was measured by spectrophotometry at 260 nm. Ingeneral, a number of centrifugation and redispersion stages of four, orless, is sufficient for achieving the desired degree of purification.

As this stage, the quantity of encapsulated iodine was determined asdescribed in example 1, by treatment of an aliquot quantity with sodiumdodecyl-sulphate (SDS): 0.1 ml of 10% aqueous SDS is added to 0.9 ml ofthe solution of liposomes, and the mixture is heated for 5 minutes at40° C., then the spectrophotometric reading is taken (the control beingan identical sample without encapsulated iodine). The optical densitycorresponding to 1 μg of iodine/ml of solution is 0.054 at 260 nm. Bymeans of a particle counter (COULTER Nanosizer), the mean size off theliposomic vesicles and also the polydispersivity thereof wereestablished.

The results appear in the following table. The tests 1 and 2 concernsamples of suspensions in a saline solution; the tests 3 concernsuspensions in glucose medium.

    ______________________________________                                              Membrane    Means size       Encapsulated                                     porosity    of vesicles                                                                             Polydis-                                                                             iodine (g/g                                Test  (μm)     (nm)      persivity                                                                            of lipids)                                 ______________________________________                                        1     initial state                                                                             610       5      2.23                                       1A      2 μm   555       5      2.49                                       2     initial state                                                                             662       5      3.02                                       2A      1 μm   482       4      3.75                                       2B    0.8 μm   468       3      3.81                                       2C    0.8 μm   346       3      3.75                                             (4 extrusions)                                                          3     initial state                                                                             919       5      3.61                                       3A    2 μm at ambient                                                            temperature 708       3      3.12                                       3B    2 μm at 75° C.                                                                  883       3      3.96                                       ______________________________________                                         *In this test, extrusion took place at ambient temperature instead of         75° C.                                                            

The results of this table show that a single extrusion operation leadsto a significant increase in the amount of encapsulated iodine and adecrease of the polydispersivity index P. In addition, the I/lproportion of encapsulated iodine (and also the degree of homogenisationof the size of the vesicles) increases when the dimension of the poresof the filtering membrane decreases.

In other tests, it has been possible to increase the encapsulated iodinecontent up to about 6 mg/mg of lipids.

EXAMPLE 3

In accordance with E. SPONTON et al. (Intern. J. Pharmaceutics (1985)23, 299), a solution was prepared which contains 543 mg of dispalmitoylphosphatidyl choline (FLUKA), 57 mg of dipalmitoyl phosphatidic acid(FLUKA) and traces of ¹⁴ C-tripalmitine (Amersham, 0.1 μCi) inchloroform (42 ml). 14 ml of this solution were placed in a 200 ml flaskand evaporated to dryness in a rotary evaporator under a partial vacuumat 25° C. There were then added 25 ml of a 61.2% solution of iopamidol(BRACCO) (corresponding to 300 mg of iodine per ml) previously heated toabout 55° C., and the mixture was allowed to incubate for two hours atambient temperature. This mixture was then subjected to fivecentrifuging operations in succession (one at 235,000 g for 30 minutes,followed by four at 29,000 g for 30 minutes at 4° C.), each of thesecentrifuging operations being followed by the residue being resuspendedin a saline solution (NaCl 0.9Tris-HCl 10 mM, pH 7.2). There wasthereafter determined, spectrophotometrically at 260 nm (see thepreceding example), the residual concentration of iopamidol in thewash-waters of the last washing operation, and also that of theencapsulated solution after rupturing of the liposomic vesicles bysodium dodecyl-sulphate (SDS). In this way, there were measured 0.08 mgof iodine per ml in the residual wahing waters, and 7.8 mg per ml ofencapsulated iodine per ml of liposome solution in the washedcomposition (25 ml). By analysis, using a scintillation counter (BeckmanLS 8100) of an aliquot quantity of the final composition (see example4), it was established that the concentration of the lipids was 5.2mg/ml (this corresponding to 65% of the initial lipids).

The preparation of the liposomes as described above was repeated so asto obtain, in total, 375 ml of suspension containing 7.7 mg ofencapsulated iodine per ml and 5.5 mg of lipids per ml (ratio ofiodine/phospholipids 1.39). This preparation was then subjected to adiafiltration at 10° C. with the aid of a microfiltration module (typeMD 020 CP2N, porosity of 0.2 μm, ENKA, Wuppertal, German FederalRepublic), the volume of liquid passing through the membrane andeliminated in the filtrate being continually replaced by addition in thesuspension of liposomes of a fresh saline solution (NaCl 0.9%, Tris-HCl10 mM pH 7.2). After elimination of 1.5 l of filtrate, the diafiltrateedsolution was concentrated, this yielding 97 ml of microfilteredliposomes, the majority of the vesicles of a size smaller than 0.2 μmhaving been eliminated. Analysis shows that this preparation contains24.9 mg of encapsulated iodine per ml and 16.4 mg of lipids per ml,i.e., a ratio of encapsulated iodine/phospholipids of 1.52. Themicrofiltration has thus made it possible to increase the encapsulatediodine/phospholipid concentration from 1.39 to 1.52, i.e. an increase ofabout 9%.

EXAMPLE 4

Solutions of liposomes (3 batch) were prepared by the technique which isdescribed in example 2 from 120 mg of lipids (NC-95H/DPPA=9/1 byweight), these lipids also containing 1.5 μCi of ¹⁴ C-tripalmitine(radioactive tracer element). Used as iodine solution (300 mg/ml) is asolution of 61.2% by weight of iopamidol in an 8 mM Tris bufferingagent, pH 7.2, 10⁻³ M disodium EDTA, this solution being filteredbeforehand through filters of 0.45 μm.

Two of the suspensions of liposomes obtained by means of the aforesaidingredients were extruded, at 75° C., through membranes whichrespectively have a porosity of 2 μm and 0.8 μm. The third solution(control) was not extruded. The three solutions, respectively labelledE-2, E-0.8 and T, were further purified by ultracentrifugation asdescribed in Example 2, then being suspended in a salt solution (0.9%NaCl, 10 mM Tris, pH 7.2); centrifugation and resuspension beingrepeated 4 times. In this way, measured by analysis as described inexample 2, there are obtained, in succession, the following respectivevalues for mean dimension of the vesicles (polydispersivity) anddcontent of encapsulated iodine in mg per mg of lipids:

T521 nm(5); 2.39

E-2 351 nm(3); 2.72

E-0.8 323 nm(2); 2.53

These liposomes were injected into the caudal vein of laboratory rats(SPRAGUE-DAWLEY) at the rate of 120 mg of iodine per kg. One hour afterinjection, the animals were killed and the blood was collected inheparinized tubes, as well as the organs, livers and lungs, which, afterhaving been dried and weighed, were burnt in an appropriate combustionapparatus (PACKARD oxidiser). The CO₂ produced by this combustion wascollected and analysed by scintillation. The blood was also analysedafter being brought into solution (aliquot quantities of 0.25 ml) in 1:1(v/v) mixture of Soluene/isopropanol (1 ml) and decolorization by H₂ O₂(0.5 ml, 32%). The various samples had added thereto 10 ml of DIMILUME(scintillation liquid) and their radioactivity was measured by means ofa BECKMANN LS-8100 scintillation counter.

The results, set out in the following table, are expressed as apercentage of the injecteed dose retained by the blood or the organsunder examination (each result is a mean of 3 measurements).

    ______________________________________                                        Specimen  Blood         Liver   Lung                                          ______________________________________                                        T         1.0           41.5    12.3                                          E-2       1.6           43.2    0.5                                           E-08      1.4           47.9    0.3                                           ______________________________________                                    

It is established, from the above results, that the homogenisation ofthe dimension of the vesicles between the size limits corresponding tothe porosity range of the 0.8 and 2 μm membrane results in aconsiderable diminution of the capturing of these vesicles by the lungs.

EXAMPLE 5

As described in example 4 (specimen E-08), suspensions of liposomes wereprepared, analysis of such suspensions having supplied the followingvalues: 32.5 mg of lipids and 70.2 mg of iodine/ml of suspension, thiscorresponding to 2.16 mg of encapsulated iodine per mg of lipids.

The next step was the injection of this suspension into four groups ofSprague-Dawley rats (five animals in each group) at the rate of 250 mgof iodine per kg.

By way of comparison, equivalent quantities of iodine, but notencapsulated in liposomes, were injected into control rats.

The animals were killed, in groups, after 30 minutes, 1 hour, 4 hoursand 24 hours and the blood was collected and conserved in heparinizedtubes. The organs (livers, spleens, kidneys and lungs) were removedbeforehand and weighed. Aliquot amounts of these organs weerehomogenized in 7 mM ammonia (5 ml) in order to determine the quantity ofresidual blood in accordance with the method described by MEIJER et al.(Clin. Chim. Acta (1962), 7, 638)

The quantity of iodine retained by the various organs referred to abovewas determined by X-ray fluorescence, using a PHILIPS PW 1410 apparatuswith a Cr anode, voltage=50 KV; current=50 mA. The corrected results forthe blood content appear in the following table and also comprisemeasurements carried out on untreated animals.

                  TABLE                                                           ______________________________________                                        μg of iodine/g of tissues                                                  Time after                                                                    injection (h)  liver  spleen  kidney                                                                              lungs                                                                              blood                                ______________________________________                                        untreated                                                                             --         0.12   0.19  0.06  0.12 --                                 animals                                                                       non-encap-                                                                            0.5        190    18    530   110  170                                sulated 1          180    11    380   48   46                                 iodine  4          90     8     33    13   1                                          24         1      1.5   0.8   1    0.2                                encapsu-                                                                              0.5        2400   5900  300   300  600                                lated   1          2700   6600  2BO   200  100                                iodine  4          2200   6200  60    160  7                                          24         800    3500  15    50   0.6                                ______________________________________                                    

The above results show how the administration of the iodine via theliposomes favours its retention by the liver and the spleen andeventually its relatively slow elimination by way of the kidneys.

In order to demonstrate the considerable technical progress which isachieved by the present invention, it is of interest to show, side byside, the results which are obtained by the invention and thoseaccording to the prior art.

For establishing this state of the art, reference is made to certain ofthe references which are cited in the introduction. The comparison inquestion is achieved by reference to the table (page 22), in which areset out a series of parameters which are inherent in the liposomesuspensions. The names of the authors of the references appear in thefirst column.

EXAMPLE 6

The liposomes which are prepared as descirbed in example 4 (specimenE-08) were injected intravenously in the dosage of 250 mg of iodine/kgto Sprague-Dawley rats which had been subjected to a computerizedtomographic examination of the liver, before and after the injection.

A Siemens Somatom 2 tomograph was used, the examination being undertakenunder the following conditions:

matrix off 256×256

field of view 14 cm

thickness of examined layer 2 mm

scanning time 5 seconds

X-rays: 125 KV mAs 230

The images were recorded before the injection of the suspension ofliposomes, then 5', 10', 15', 30', 60', 90', 2 h, 3 h and 4 h after theinjection, and there was observed an increase in contrast of the liver,expressed in Hounsfield Units (HU), this increase being between 50% and130% during the period which is between 30 minutes and 4 hours.

It should be noted that if, in the techniques disclosed in the previousexamples, dicetyl-phosphate (DCP) or dipalmitoyl-phosphatidyl glycerol(DPPG) is used in place of DPPA for preparing the liposome vesicles,similar results are experienced.

                  TABLE                                                           ______________________________________                                        Properties of liposomes containing opacifiers to X-rays                                                      Ratio by                                                                              Encap-                                                      Opacifying                                                                              weight  sulated                                                     agent     iodine/ volume                                 Inventors Lipids     (mgl.sub.2 /ml)                                                                         lipid (g/g)                                                                           (ml/g)                                 ______________________________________                                        HAVRON    Soya lecithin                                                                            Diatri-   0.14    0.4                                    et al.    cholesterol                                                                              zoate                                                              stearyl amine                                                                            (370)                                                    RYAN(1)   Phosphatidyl                                                                             Diatri-   0.9     6.2                                    et al.    choline/   zoate                                                              cholesterol                                                                              (146)                                                    ZELTZER   Lecithin/  Iosefa-   --      --                                     et al.    cholesterol/                                                                             mate                                                               stearyl amine                                                       RYAN(2)   Phosphatidyl                                                                             Diatri-   0.4     1                                      et al.    choline/   zoate                                                              cholesterol                                                         ROZENBERG --         --        1.5     --                                     et al.                                                                        BENITA    Soya lecithin/                                                                           --        0.2     2.7                                    et al.    cholesterol                                                         CHENG     Egg lecithin/                                                                            Iohexol   0.25    0.6                                    et al.    phosphatidyl                                                                             (+others)                                                          glycerol/                                                                     cholesterol                                                         ZALUTSKY  Lecithin/  Diatri-   --      --                                     et al.    cholesterol/                                                                             zoate or                                                           stearyl amine                                                                            iotrol                                                   INVENTION NC 95 H/   Iopamidol 3-4     7-12                                   (Example  DPPA       (300)                                                    type)     (soya leci-                                                                   thin)                                                               ______________________________________                                    

We claim:
 1. An injectable aqueous composition, developed for opacifyingorgans for X-ray examination, formed of a suspension in a physicallytolerable aqueous medium of liposomic vesicles having a lipidic membranecontaining encapsulated in these vesicles, at least one iodinatedorganic compound opaque to X-rays in aqueous solution, wherein thevesicles of the liposomes have a mean size which is between 0.15 and 3μm, and that the ratio of the weight of the iodine encapsulated in theliposomic vesicles to the weight of the lipids of the said membrane isfrom 1.75-6):1.
 2. An injectable composition according to claim 1,wherein the polydispersivity of the size of the vesicles in the rangeunder consideration is not higher than
 4. 3. An injectable compositionaccording to claim 1, of which the concentration of lipids in suspensionin the said aqueous medium is between 20 and 40 g/l, wherein itsviscosity does not exceed 30 mP.s.
 4. An injectable compositionaccording to claim 1, wherein the iodinated organic compound is selectedfrom Iopamidol, Iomeprol, Iohexol, Iopentol, Iopromide, Iosimide,Ioversol, Iotrolan, Iotasul, Iodixanol, Iodecimol,1,3-bis-(N-3,5-bis-(2,3-dihydroxypropylaminocarbonyl)-2,4,6-triiodo-phenyl)-N-hydroxyacetyl-amino)-propane.5. An injectable composition according to claim 1, wherein the lipidsare selected from one or more of the hydrogenated soya lecithins,dipalmitoyl-phosphatidyl choline (DPPC), distearoyl-phosphatidyl choline(DSFC), sphingomyeline (SM), dicetyl-phosphate (DCP),diplamitoyl-phosphatidyl glycerol (DPPG) and dipalmitoyl-phosphatidicacid (DPPA).
 6. An injectable composition according to claim 1 whereinsaid aqueous medium exterior to said liposomes contains no more than 0.2to 2 mg/ml of an iodinated organic compound.